Melanotan-II: Molecular Structure and Chemistry

Molecular Formula and Basic Properties

Melanotan-II is a synthetic cyclic heptapeptide with the molecular formula C₅₀H₆₉N₁₅O₉ and a molecular weight of 1,024.18 Da. This relatively small size for a peptide (compared to proteins which can be hundreds of thousands of Daltons) allows it to cross biological membranes more easily than larger peptides, contributing to its bioavailability and ability to reach target tissues including the brain.

Amino Acid Sequence

The linear amino acid sequence of MT-II, before cyclization, is:

Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂

Breaking this down:

• Ac- = N-terminal acetylation (acetyl group attached to amino terminus)
• Nle = Norleucine (non-standard amino acid, isomer of leucine)
• cyclo[...] = Indicates cyclic structure formed by lactam bridge
• Asp = Aspartic acid (forms one end of lactam bridge)
• His = Histidine
• D-Phe = D-Phenylalanine (unnatural D-configuration)
• Arg = Arginine
• Trp = Tryptophan
• Lys = Lysine (forms other end of lactam bridge)
• -NH₂ = C-terminal amidation (amide group at carboxyl terminus)

The cyclic structure is created through a lactam (amide) bond between the side chain carboxyl group of aspartic acid (position 3) and the side chain amino group of lysine (position 7). This creates a 23-membered ring that constrains the peptide into a specific three-dimensional conformation.

Key Structural Features

Several structural features distinguish MT-II from natural peptides and contribute to its unique properties:

1. Cyclic Structure

The lactam bridge creates a rigid, constrained structure that "pre-organizes" the peptide into the optimal conformation for binding melanocortin receptors. This eliminates the entropic cost of adopting the correct conformation upon binding, increasing binding affinity. The cyclic structure also provides resistance to exopeptidases (enzymes that cleave peptides from the ends), dramatically extending half-life.

2. D-Phenylalanine

The inclusion of D-phenylalanine (position 4) is crucial for stability. Natural peptidases are evolved to cleave bonds between L-amino acids and have difficulty recognizing and cleaving bonds involving D-amino acids. This single modification provides significant resistance to enzymatic degradation. The D-configuration also affects the peptide's three-dimensional structure, contributing to receptor binding properties.

3. Norleucine

Norleucine (Nle) at position 1 is a non-standard amino acid—an isomer of leucine with a straight-chain rather than branched side chain. This substitution was made to improve stability (norleucine is less susceptible to oxidation than methionine, which it replaced from earlier analogs) while maintaining similar hydrophobicity and size.

4. Terminal Modifications

Both N-terminal acetylation (Ac-) and C-terminal amidation (-NH₂) protect the peptide from exopeptidases that would otherwise rapidly degrade it from the ends. These modifications are common in bioactive peptides and dramatically extend half-life.

5. Core Pharmacophore

The sequence His-D-Phe-Arg-Trp (positions 3-6) represents the "pharmacophore"—the minimal structural unit required for melanocortin receptor binding. This tetrapeptide sequence is derived from the natural α-MSH sequence and is essential for biological activity. Modifications to these residues typically abolish or dramatically reduce activity.

Three-Dimensional Structure and Conformation

Conformational Constraint

The cyclic structure of MT-II constrains it into a relatively rigid three-dimensional shape. Nuclear magnetic resonance (NMR) spectroscopy and computational modeling studies have revealed that MT-II adopts a β-turn conformation, with the pharmacophore residues (His-D-Phe-Arg-Trp) positioned on one face of the molecule in an orientation optimal for receptor binding.

This conformational constraint is key to MT-II's high potency. Linear peptides like natural α-MSH exist as flexible molecules that can adopt many different conformations. When binding to a receptor, they must "find" the correct conformation, which has an entropic cost (loss of conformational freedom). MT-II, being pre-organized into the correct shape, doesn't pay this entropic penalty, resulting in tighter binding and higher potency.

Receptor Binding Interactions

The interaction between MT-II and melanocortin receptors involves multiple types of molecular interactions:

Electrostatic interactions: The positively charged arginine (Arg) side chain forms salt bridges with negatively charged residues in the receptor binding pocket. This is a key anchor point for binding.

Hydrogen bonding: The histidine (His) imidazole ring and backbone amide groups form hydrogen bonds with receptor residues, contributing to binding affinity and specificity.

Hydrophobic interactions: The aromatic side chains of phenylalanine (Phe) and tryptophan (Trp) insert into hydrophobic pockets in the receptor, providing significant binding energy through van der Waals forces.

Shape complementarity: The overall three-dimensional shape of MT-II complements the receptor binding pocket, maximizing contact surface area and binding affinity.

The combination of these interactions results in binding affinities in the nanomolar range (Kd ~ 1-10 nM for MC1R, MC3R, MC4R), approximately 1,000-fold tighter than natural α-MSH.

Conformational Flexibility

While MT-II is more rigid than linear peptides, it retains some conformational flexibility. The cyclic structure can undergo limited conformational changes, particularly in the loop region opposite the pharmacophore. This flexibility may allow MT-II to adapt slightly to different melanocortin receptor subtypes, contributing to its non-selective binding profile (activates MC1R, MC3R, MC4R, and MC5R with similar potencies).

Physicochemical Properties

Solubility

MT-II is moderately water-soluble due to the presence of charged and polar amino acids (Asp, His, Arg, Lys) that can interact with water molecules. The peptide readily dissolves in water, saline, or bacteriostatic water at concentrations up to 10-20 mg/mL, which is sufficient for practical use.

Solubility is pH-dependent:

• Acidic pH (3-5): Good solubility; histidine and arginine are protonated and positively charged
• Neutral pH (6-8): Good solubility; optimal for biological activity and stability
• Basic pH (>8): Reduced solubility; deprotonation of histidine reduces net charge

The peptide is poorly soluble in organic solvents (ethanol, acetonitrile, DMSO) except at low concentrations. This aqueous solubility is advantageous for formulation and administration but limits options for alternative delivery routes.

Stability

MT-II's stability is influenced by multiple factors:

Temperature stability:

• Frozen (-20°C or below): Stable for years in lyophilized form
• Refrigerated (2-8°C): Stable for months to years (lyophilized) or weeks (reconstituted)
• Room temperature (20-25°C): Gradual degradation over days to weeks
• Elevated temperature (>30°C): Rapid degradation, particularly in solution

pH stability:

• Acidic pH (<4): Risk of acid-catalyzed hydrolysis of peptide bonds
• Neutral pH (5-8): Optimal stability
• Basic pH (>9): Risk of base-catalyzed hydrolysis and racemization

Oxidation susceptibility:

The tryptophan residue is particularly susceptible to oxidation, which can occur through exposure to oxygen, light (especially UV), or oxidizing agents. Oxidized tryptophan (oxindolylalanine or other products) reduces biological activity and may create potentially harmful degradation products. Protection from light and oxygen (through proper storage and formulation) is essential.

Aggregation:

Peptides can aggregate (clump together) through hydrophobic interactions, hydrogen bonding, or disulfide bond formation. MT-II is relatively resistant to aggregation due to its cyclic structure and charged residues, but aggregation can occur at high concentrations, elevated temperatures, or with repeated freeze-thaw cycles. Aggregated peptides have reduced biological activity and may cause injection site reactions.

Partition Coefficient (LogP)

The octanol-water partition coefficient (LogP) is a measure of lipophilicity (fat-solubility). MT-II has a LogP of approximately -1 to 0, indicating it is moderately hydrophilic (water-preferring) but with some lipophilic character from the aromatic amino acids. This balanced lipophilicity allows MT-II to:

• Dissolve in aqueous solutions for injection
• Cross biological membranes (including blood-brain barrier) to some degree
• Distribute to both aqueous and lipid compartments in the body

The moderate lipophilicity contributes to MT-II's pharmacokinetic properties, including its ability to reach CNS targets for sexual and appetite effects.

Ionization and Charge State

At physiological pH (7.4), MT-II carries a net positive charge due to:

• Positively charged residues: Arginine (always positive), Lysine (positive at pH 7.4), Histidine (partially positive at pH 7.4)
• Negatively charged residues: Aspartic acid (negative at pH 7.4)
• Terminal modifications: N-terminal acetylation removes positive charge; C-terminal amidation removes negative charge

The net positive charge (approximately +2 to +3 at pH 7.4) affects MT-II's interactions with cell membranes, receptors, and other biological molecules. It also influences solubility, stability, and pharmacokinetics.

Chemical Reactivity and Degradation Pathways

Hydrolysis

Peptide bonds can be cleaved by water (hydrolysis), particularly under acidic or basic conditions or in the presence of enzymes (peptidases). MT-II's cyclic structure and D-amino acid provide significant protection against enzymatic hydrolysis, but chemical hydrolysis can still occur:

• Acid-catalyzed hydrolysis: Occurs at pH <4, particularly at elevated temperatures. Aspartic acid residues are especially susceptible.
• Base-catalyzed hydrolysis: Occurs at pH >9. Can also cause racemization (conversion of L-amino acids to D-amino acids or vice versa).
• Enzymatic hydrolysis: Peptidases in blood and tissues can slowly cleave MT-II, though the cyclic structure and D-amino acid provide resistance. This is the primary elimination pathway in vivo.

Oxidation

Oxidation is a major degradation pathway for MT-II, primarily affecting the tryptophan residue:

Tryptophan oxidation: Can produce multiple oxidation products including N-formylkynurenine, kynurenine, hydroxytryptophan, and oxindolylalanine. These modifications reduce or eliminate biological activity.

Oxidation triggers:

• Exposure to oxygen (air)
• UV or visible light
• Metal ions (iron, copper)
• Peroxides or other oxidizing agents
• Elevated temperature

Protection strategies:

• Store in dark containers or wrap in foil
• Minimize air exposure (use nitrogen or argon atmosphere for long-term storage)
• Add antioxidants (though this may affect biological activity)
• Keep refrigerated or frozen
• Use fresh solutions rather than storing reconstituted peptide long-term

Deamidation

Asparagine and glutamine residues can undergo deamidation (loss of ammonia group), converting to aspartic acid and glutamic acid respectively. While MT-II doesn't contain asparagine or glutamine, the C-terminal amide can be hydrolyzed to a carboxylic acid, reducing biological activity. This is generally a slow process but can be accelerated by elevated pH or temperature.

Racemization

Under basic conditions or elevated temperatures, L-amino acids can partially convert to D-amino acids (or vice versa). This creates diastereomers with potentially different biological activities. The D-phenylalanine in MT-II is intentional, but racemization of other residues would create unwanted isomers. Racemization is generally slow under proper storage conditions but can occur during synthesis if conditions aren't carefully controlled.

Structure-Activity Relationships (SAR)

Essential Residues

Structure-activity relationship studies have identified which parts of MT-II are essential for biological activity:

His-D-Phe-Arg-Trp pharmacophore: This tetrapeptide sequence is absolutely required for melanocortin receptor binding. Modifications to any of these residues typically abolish or dramatically reduce activity:

• Histidine (His): The imidazole ring is critical for receptor binding. Replacement with other amino acids eliminates activity.
• D-Phenylalanine (D-Phe): The aromatic ring and D-configuration are both important. L-Phe has reduced activity; non-aromatic amino acids eliminate activity.
• Arginine (Arg): The positively charged guanidinium group is essential. Replacement with lysine (also positive) maintains some activity but is less potent.
• Tryptophan (Trp): The indole ring is critical. Replacement with phenylalanine (smaller aromatic) reduces activity; non-aromatic amino acids eliminate activity.

Cyclic structure: The lactam bridge is crucial for high potency. Linear analogs of MT-II have 10-100 fold lower potency, demonstrating the importance of conformational constraint.

D-amino acid: The D-phenylalanine provides both stability (resistance to peptidases) and optimal receptor binding geometry. L-phenylalanine analogs have reduced potency and much shorter half-lives.

Modifiable Positions

Some positions in MT-II can be modified without completely eliminating activity:

Position 1 (Norleucine): Can be replaced with other hydrophobic amino acids (leucine, methionine, etc.) with modest effects on potency. The choice of norleucine was made for stability rather than potency.

Position 2 (Aspartic acid): Required for lactam bridge formation but the exact position of the bridge can be varied. Different cyclization strategies have been explored in analog development.

Position 7 (Lysine): Also required for lactam bridge. Can potentially be replaced with ornithine (shorter chain) or other diamino acids.

Terminal modifications: N-terminal acetylation and C-terminal amidation improve stability but aren't absolutely required for receptor binding. However, they dramatically affect pharmacokinetics.

Receptor Selectivity

MT-II is relatively non-selective, activating MC1R, MC3R, MC4R, and MC5R with similar potencies (nanomolar range). This lack of selectivity is both an advantage (multiple effects from single compound) and disadvantage (side effects from unintended receptor activation).

Efforts to create selective analogs have focused on modifying residues outside the core pharmacophore or changing the cyclic structure. For example:

• Afamelanotide (Melanotan-I): Linear structure, selective for MC1R
• Bremelanotide (PT-141): Modified from MT-II, reduced tanning effects while maintaining sexual effects
• Setmelanotide: Selective MC4R agonist for obesity treatment

These examples demonstrate that receptor selectivity can be achieved through structural modifications, though often at the cost of reduced overall potency or altered pharmacokinetics.

Analytical Characterization

Methods for Confirming Identity and Purity

Multiple analytical techniques are used to characterize MT-II:

Mass Spectrometry (MS):

• Confirms molecular weight (1,024.18 Da)
• Detects oxidation, deamidation, or other modifications
• Can identify impurities and degradation products
• High-resolution MS provides elemental composition

High-Performance Liquid Chromatography (HPLC):

• Separates MT-II from impurities based on hydrophobicity
• Provides purity percentage (area under curve analysis)
• Can detect deletion sequences, truncated peptides, diastereomers
• Retention time serves as identity confirmation

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Provides detailed structural information
• Confirms amino acid sequence and modifications
• Reveals three-dimensional conformation
• Can detect impurities and degradation products
• Requires larger sample amounts and specialized equipment

Amino Acid Analysis:

• Confirms amino acid composition and ratios
• Detects substitutions or deletions
• Provides quantitative assessment of peptide content
• Requires complete hydrolysis of peptide bonds

Circular Dichroism (CD) Spectroscopy:

• Provides information about secondary structure
• Can detect conformational changes or misfolding
• Useful for assessing stability and aggregation

Spectroscopic Properties

MT-II has characteristic spectroscopic properties that can be used for identification and quantification:

UV-Visible Absorption:

• Maximum absorption around 280 nm (from tryptophan and phenylalanine)
• Can be used for concentration determination (Beer-Lambert law)
• Oxidation of tryptophan changes absorption spectrum

Fluorescence:

• Tryptophan fluorescence (excitation ~280 nm, emission ~350 nm)
• Sensitive to local environment and conformational changes
• Can be used to study receptor binding and conformational dynamics